TABLE 2.
Kinetic parameters of Gly mutants
NcLAD and HjLAD NAD+ and l-arabinitol were used as cofactor and substrate, respectively. ReXDH NAD+ and xylitol were used as cofactor and substrate, respectively. HmGDH NADP+ and glucose were used as cofactor and substrate, respectively. Results are the means ± S.D. from three experiments. ND, not determined due to the very low activity of the enzyme.
| Enzymes | Specific activity |
Km |
kcat | kcat/Km | |
|---|---|---|---|---|---|
| Coenzyme | Substrate | ||||
| Units mg-protein−1 | μm | mm | min−1 | mm−1min−1 | |
| NcLADWT | 41.2 ± 4.1 | 0.90 ± 0.02 | 16.3 ± 1.2 | 1600 ± 39 | 100 ± 9 |
| G77A | 0.400 ± 0.02 | 0.020 ± 0.01 | 31.1 ± 2.3 | 15.8 ± 2.2 | 0.500 ± 0.09 |
| G77S | 0.300 ± 0.02 | 0.020 ± 0.01 | 72.4 ± 6.2 | 12.1 ± 2.1 | 0.160 ± 0.04 |
| G77E | ND | ND | ND | ND | ND |
| G77D | ND | ND | ND | ND | ND |
| G77C | ND | ND | ND | ND | ND |
| G77H | ND | ND | ND | ND | ND |
| HjLADWT | 89.2 ± 10.3 | 0.20 ± 0.01 | 16.6 ± 1.3 | 3560 ± 70 | 222 ± 18 |
| G90A | 0.300 ± 0.02 | 1.5 ± 0.1 | 38.1 ± 3.1 | 12.3 ± 2.1 | 0.750 ± 0.18 |
| G90E | ND | ND | ND | ND | ND |
| ReXDHWT | 32.1 ± 5.7 | 0.50 ± 0.01 | 15.5 ± 3.1 | 1220 ± 28 | 78.0 ± 16.9 |
| G65E | ND | ND | ND | ND | ND |
| HmGDHWT | 38.4 ± 7.6 | 0.50 ± 0.01 | 12.3 ± 1.6 | 1900 ± 54 | 158 ± 30 |
| G61E | ND | ND | ND | ND | ND |